Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

ABSTRACT

A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C 1  and C x  types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

CONTRACT ORIGIN OF THE INVENTION

The United States Government has rights in this invention pursuant tocontract No. DE AC02-83CH10093 between the United states Department ofEnergy and the Midwest Research Institute.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to a purification process forAcidothermus cellulolyticus endoglucanases, and pertains morespecifically to a purification protocol used to purify the high and twolow molecular weight endoglucanases from the cellulase enzyme complexobtained from Acidothermus cellulolyticus bacterium, which has beensubmitted to the American Type Culture Collection (ATCC) undercollection number 43068.

2. Description of the Prior Art

Cellulose consists of long insoluble chains of covalently bonded glucosemolecules, and in this condition, these long insoluble chains are toolarge to be transported through human and animal cell walls. However,through the agency of microorganisms, such as fungi and bacteria,enzymes known as cellulases are secreted, and these enzymes hydrolyze ordepolymerize the cellulose into its monomeric component of glucose,which is a sugar that can be readily transported through the cell walland metabolized.

Cellulases are enzyme complexes that include three different types ofenzymes. These enzymes are endoglucanase, exoglucanase and a cellobiase,and the synergistic action of these three enzymes are necessary tocompletely depolymerize cellulose into glucose.

The synergistic reaction occurs as a result of a sequential, cooperativeaction between the three enzyme components in a complex in which theproduct of one enzyme reaction becomes the substrate for the nextenzyme.

While many microorganisms have been classified as cellulolytic, only afew possess a complete cellulase complex capable of efficientdeploymerization of crystalline cellulose under certain conditions.Trichoderma reesei (Simmons, E.G., 1977. Abst, Ind Mycol. Cong., p.618), Which is a common soil fungus of the Deuteromycete family, is onesuch cellulolytic microorganism; however, the percent maximum activityper (mM) of cellobiase is too limited and this cellulase is not highlystable when exposed to high temperature ranges. Although the enzymes ofthe organism Trichoderma reesei have been subjected to intensivebiochemical study (Shoemaker, S.P. and R.D., Brown, 1978. Enzymicactivities of endo-1 4-β-D Glucanases EC-3.2.1.4 purified from T.viride. Biochim, Biophys. Acta. 523,1, pp. 133-146.), they have not beenshown to be stable in the presence of high temperatures. For example, awild type of Trichoderma reesei is known to secrete cellulase enzymeswith optimal temperatures of 45° C. for total activity, 55° C. forendoglucanase activity, and after about one hour at 60° C., thecellulase from this Trichoderma reesei looses half of its totalactivity. Cellulases produced by various organisms have been describedin U.S. Pat. Nos. 3,232,832 (Rhizophus), 3,398,055 (Trichoderma virideATCC 16425; and other fungi); 3,438,864 (Eumyees mold organism ATCC16425, 3,677,899 (Lampteromyces or Formitopsis); and 3,734,831. However,cellulase preparations from Trichoderma viride, while known to be higherin C_(l) enzyme activity than other commercially available cellulases,have the drawback that optimum temperature for their enzymic reactionsis about 50° C. (Mandels, et al. "Cellulases and Their Applications",ACS. Vol. 95, pp 398-411 (1969)).

U.S. Pat. No. 4,081,328 discloses a thermostable cellulase exhibitingboth C_(l) and C_(x) types of cellylytic activities produced by thefungal organism Thiolavia terestris (NRRL 8126). The thermostablecellulase preparation exhibits C_(l) and C_(x) cellulytic activities attemperatures from about 60° to about 70° C. (140° to 160° F.).

At present, mesophilic cellulases with temperature optima near 45° to50° C. from Trichoderma species (Mandels and Weber, 1969. "Production ofCellulases", Advan. Chem. Ser., Vol. 95, pp. 391-414), are usedindustrially, however, industrial use of mesophilic cellulases requireexpensive refrigeration to cool the process stream and the chances ofcontamination by opportunistic microorganisms are greatly increased.

Therefore, a need exist to provide a higher optimum temperature rangecellulase enzyme that is capable of replacing the lower optimumtemperature (60° to 70° C.) mesophilic cellulase enzymes.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, it has been foundthat the higher optimum temperature (70° to 80° C.) cellulase enzymecomplex from the bacterium Acidothermus cellulolyticus produced by theprocess of the invention can be used to replace the mesophilic cellulaseenzymes that are currently used industrially.

The invention process for the preparation of the high optimumtemperature cellulase enzymes provides means for concentrating theculture broth of Acidothermus cellulolyticus by using ammonium sulfateprecipitation (between 40 to 60% saturated solutions) or byultrafiltration using an Amicon ultrafiltration apparatus equipped withPM-lo membranes and the concentrate can be stored at -20° C. in thepresence of 20% glycerol for periods greater than a year with no loss inenzyme activity.

In accordance with the second aspect of the invention, the cellulaseenzyme complexes produced by Acidothermus cellulolyticus possesses bothC₁ and C_(x) enzyme activitity, and exhibit cellulolytic activity attemperatures ranging from 20 to 110° C. and at pH's ranging from 2 to 9,preferably at pH's from about 4 to about 8; and most preferably at a pHof about 5.

An important aspect of the cellulase of the invention is thethermotolerant characteristics where the enzyme exhibits a highstability upon exposure to heat. After incubating the crude enzymesolution for about two hours at 90° C., 20% of enzyme activity stillremains intact.

The invention is further directed to the purification andcharacterization of three thermostable endoglucanases produce by thethermophilic bacterium Acidothermus cellulolyticus. The high molecularweight endoglucanase appears to be less table with a temperature optimumnear 65° C., while of the two low molecular weight endoglucanases, Endo1 has a temperature optimum near 80° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the growth cycle of Acidothermus cellulolyticus grown on5 (g/l) cellobiase in a 5 l. stirred tank reactor at 55° C. at a pH ofabout 5.2.

FIG. 2 depicts the activity temperature profile for the crude enzymefrom A. cellulolyticus.

FIG. 3 depicts the activity incubation pH profile for the crude enzymefrom A. cellulolyticus.

FIG. 4 depicts the inhibition of Acidothermus FP (filter paper) activityby inorganic and organic ions.

FIG. 5 depicts a comparison of the inhibition of the FP (filter paper)activity of A. cellulolyticus by the end-product, cellobiose, withTrichoderma Reesei (Rut C-30) and Clostridium thermocellum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Acidothermus cellulolyticus has been studied in detail taxonomicallyresulting in both a new genus and species name to support the criteriafor novelty of the organism and the cellulase secreted by this organisminto the medium in which it has been grown. The detailed characteristicsof the organism is published in the International Journal of SystematicBacterioloy, 36, 1986 pp. 435-443. The microorganism utilizes a widevariety of simple sugars, as Well as a variety of cellulosic materialssuch as cellulose powder, as the sole carbon source. A minimal mineralmedia containing inexpensive and readily obtainable salts and a nitrogensource (inorganic ammonium salts or organic nitrogenous materials suchas amino acids) is used. Yeast extract may be used by supplementationinto the minimal mineral media to serve as a source of various growthfactors.

This bacterium grows within a pH range of 3 to 7 and a temperature rangeof 40° to 70° C. with the optimum growth conditions near pH 5 and atemperature of 55° C.

Stock cultures of Acidothermus cellulolyticus are stored at about -70°C. with the addition of dimethylsulfoxide (DMSO) to the primary culturebroth. For the cultivation of Acidothermus cellulolyticus for theproduction of cellulase, primary culture inocula are started by thawingthe frozen cultures that have been stored and transferring them toflasks containing sterile culture media. The primary culture inocula areused to start the inocula for larger fermenters with the volume of theinocula equal to one tenth the volume of the fermenters to beinoculated. The initial PH of the culture media is about 5.2 and theincubation temperature is set to 55° C. This microorganism reachesmaximum growth in about 20 to about 36 hours after which secretion ofthe cellulase enzyme complex is found to occur.

Crude culture broths from this bacterium show optimal temperature forthe depolymerization of crystalline cellulose 75° C., and 83° C. isfound for endoglucanase carboxymethyl cellulose (CMC) activity. At 90°C., 38% of optimal activity for degradation of cellulose is found, and60% of endoglucanase activity is found under standard assay conditions.This cellulase enzyme can replace the mesophilic cellulase enzymes usedindustrially since its optimum temperature is between 70° to 80° C.,above the 60° to 70° (140° to 160° F.) pasteurization range currentlyused by industry.

EXAMPLE 1

Stock cultures of Acidothermus cellulolyticus were maintained at -70° C.by the addition of 77 microliters of DMSO to 1 ml of culture grown toearly stationary phase. One milliliter of stock culture was thawed andtransferred to a 50 ml shake flask containing 20 ml medium With thecompostion shown in Table 1. The flask is incubated at 55° C. for 20 to36 hours and the culture transferred to a 500 ml flask containing 200 mlof culture media.

This flask is used as the inoculum to a 5-liter fermenter with a workingvolume of 2 liters. The pH is controlled at 5.2 by titration with KOH(1N) or HCl (1N). The enzyme activities are measured over 48 hours ofthis fermentation cycle. One ml samples are taken and centrifuged at12,000xg with 0.5 ml of the supernatant being used to measure the filterpaper (FP) activity in accordance to the method recommended by theInternational Union of pure and Applied Chemistry (lUpAC), 1987, and thesame is incorporated herein by reference in its entirety. In this method0.5 ml of the supernatant is mixed with 0.75 ml citrate buffer (0.1 M),pH 4.8, to which 0.25 ml of deionized (Dl) water is added to bring thetotal volume in the assay mixture to 1.5 ml. The assay tubes containing50 mg strips of Whatman #1 filter paper are preincubated at 55° C.before the addition of the enzyme solution, followed by incubation at55° C. for one hour. The reducing sugar released by the action of theenzyme is measured using the 3,5-dinitrosalicylic acid (DNS) reagent andmethod. A standard curve by which to compare the glucose released isconstructed using accurately weighed glucose standards within the rangeof 0 to 5 mg. The amount of enzyme added was adjusted to give a totalrelease of glucose at the end of a one hour incubation period equal to 2mg. The total activity obtained during the course of the fermentation is0.02 international filter paper units per ml (IFPU/ml). This low valuefor enzyme activity is natural with many Wild type organisms. Strainimprovements are required to increase the cellulase enzyme titer andtotal productivity. The growth cycle is shown in FIG. 1.

                  TABLE 1                                                         ______________________________________                                        Media Composition for Growing Acidothermus                                    celluolyticus                                                                 Component     g/L in deionized water                                          ______________________________________                                        NH.sub.4 Cl   2.0                                                             NH.sub.2 PO.sub.4                                                                           2.0                                                             Na.sub.2 HPO4 0.2                                                             MgSO.sub.4 7H.sub.2 O                                                                       0.4                                                             CaCl.sub.2     0.04                                                           Yeast Extract 1.0                                                             Carbon source 5.0                                                             FeSO.sub.4     0.01                                                           pH adjusted to 5.2                                                            ______________________________________                                    

EXAMPLE 2

The 120 liter fermenter broth is centrifuged using a CEPA type 41continuous centrifuge followed by concentration of the supernatant usingan Amicon DC-30 ultrafiltration apparatus equipped with pM-lo hollowfiber filters to a level of 2 IFPU/ml. The concentrated enzyme solutionis mixed with 20% glycerol and stored frozen at -20° C.

EXAMPLE 3

The temperature range over which the enzymes secreted by Acidothermuscellulolyticus is examined and the results are depicted in FIG. 2. Thetemperature range for filter paper (FP) as well as the measuredendoglucanase (CMC) activity is shown over the range of 30° to 110° C.The activities are measured according to the lUPAC methodology publishedin the reference given in Example 1. The broad temperature range overwhich the enzymes have significant activity is clearly evident. Thetemperature optimum at pH 5.0 for filter paper actively is near 75° C.and the endoglucanase activity near 83° C.

EXAMPLE 4

The pH range over which the enzymes secreted by Acidothermuscellulolyticus are active is shown in FIG. 3. Filter paper (FP) activityand endoglucanase (CMC) activity is measured over the range of about 2.2to about 8 using a constant ionic strength buffer system at about 65° C.

EXAMPLE 5

The inhibition of the filter paper (FP) activity by both inorganic andorganic ions is shown in FIG. 4. In this figure the activity curve shownversus the concentration of salt is measured according to the lUPAcmethodology discussed in Example 1, except that various amounts of aconcentrated 1 M NaCl solution is added so that the final concentrationof NaCl equaled that shown by the data points. The organic ionconcentration is varied in a similar manner by the addition of 1 Macetate buffer (pH 5) up to the indicated concentrations. The observedfilter paper activity decreased 25% in the presence of highconcentrations of inorganic ions (salt). The observed filter paperactivity is more sensitive to the presence of organic ions (acetate) asshown by the activity decreasing to 40% of maximum at acetate levelsnear 0.5 M.

EXAMPLE 6

The inhibition of filter paper (FP) activity by the end productinhibitor cellobiose is shown in FIG. 5. Cellobiose is a potentinhibitor of the cellulases secreted by Trichoderma reesei with completeinhibition occuring at approximately 60 mM. The cellulase fromAcidothermus cellulolyticus is much more resistant to inhibition bycellobiose with activity observed at levels near 0.5 M.

EXAMPLE 7 High Molecular Weight Endoglucanase

The high molecular weight endoglucanase is purified from the crudeculture broth in the following sequence of steps. First, the crudeculture broth from a 150 liter fermenter is centrifuged at 20,000 rpm ina CEPA continuous flow, bowl type centrifuge to remove the Acidothermuscellulolyticus microorganism. The supernatant recovered followingcentrifugation is concentrated greater than ten fold using an AmiconDC-30 ultrafiltration apparatus. Aliquots of the concentrated stock arefurther concentrated using a much smaller Amicon stirred cellultrafiltration apparatus equipped with PM-10 membrane filters beforeloading onto a semi-preparative size exclusion chromotography (SEC)column. The SEC column is manufactured by Toyo Soda (TSK 3000 SWG) andmeasured 21.5 by 700 mm. The proteins elute from this column accordingto their molecular weight and are fractionated and collected asfractions of peaks. The molecular weight of these enzymes are found torange from about 156,600 to about 203,400 daltons.

The fractions containing the high molecular weight endoglucanase peakfrom this column are pooled and diafiltered against 15 mM Bis-Tris pH6.5 ion exchange chromatography (IEC) loading buffer. The high molecularweight endoglucanase is then loaded onto a Pharmacia (Mono Q) IECcolumn.

Following washing with ten column volumes of loading buffer, the highmolecular endoglucanase is eluted off the IEC using a shallow gradientof increasing ionic strength buffer that increases linearly from 0 to 50mM in Nacl in 320 minutes at a flow rate of 1 ml per minute. Thepurified high molecular weight endoglucanase is eluted from this columnat a gradient ionic strength corresponding to approximately 25 mM inNaCl.

The following diagram is a schematic representation of the purificationprotocol for the high molecular weight endoglucanase from Acidothermuscellulolyticus. ##STR1##

EXAMPLES 8 and 9

The low molecular weight endoglucanases 1 and 11 are purified from thecrude culture broth of a 150-liter fermentation in the followingsequence of steps.

First, the crude culture broth from the 150 liter fermenter iscentrifuged at 20,000 rpm in a CEPA continuous flow bowl type centrifugeto remove the Acidothermus cellulolyticus microorganism. The supernatantrecovered following centrifugation is concentrated at least ten foldusing an Amicon DC-30 ultrafiltration apparatus. Aliquots of theconcentrated stock are further concentrated using a smaller Amiconstirred ultrafiltration apparatus equipped with a PM-lO membrane filterbefore loading onto a semi-preparative size exclusion chromotography(SEC) column. The low molecular weight endoglucanase peak eluting fromthe TSK 3000 SWG SEC column are fractionated with the activity pooledand ultraconcentrated to 10 ml. The ultraconcentrate is divided into sixequal fractions to which 6.200 grams of CsC (ultrapure) is added toeach. Concentrated acetate buffer (1 M, pH 5.0) is added (1.13 ml) andthe tubes are brought to 11.3 ml by adding water until the total weightadded is 15.82 grams. Each of the six tubes are placed in a bucket in aBeckman SW-41 swinging bucket rotor and the loaded rotor placed in aBeckman ultracentrifuge. Following centrifugation for at least 96 hoursat 35,000 rpm, the tubes are removed and fractionated. The isopycnicbanded protein is pooled from the fractions obtained followingcentrifugation, diafiltered against loading buffer, and loaded onto anion-exchange column. The Mono Q strong anion exchange column (Pharmacia)is equillbrated against 15 mM Bis-Tris pH 7.0 loading buffer before thediafiltered low molecular weight endoglucanase peak is loaded. Followingwashing with ten column volumes of loading buffer, the low molecularweight endoglucanase I and II are eluted from the column using a shallowgradient of increasing ionic strength buffer. The gradient increaseslinearly from 0 to 50 mM (Nacl concentration) in 320 minutes at a flowrate of 1 ml per minute. Low molecular weight endoglucanase I elutedfrom this column at a gradient ionic strength of approximately 25 mMwhile the low molecular weight endoglucanase 11 eluted at approximately31 mM in NaCl. The pooled low molecular weight endoglucanase I and IIpeaks are subsequently ultraconcentrated and loaded separately onto theTSK 3000 SWG SEC column and fractionated. The fractions containingendoglucanase activity are pooled and ultraconcentrated (PM-lO) toobtain the final purified low molecular weight endoglucanases I and II,which have been found to have molecular weights respectively of betweenabout 57,420 to 74,580 daltons and between about 50,000 to about 70,000daltons.

The following diagram is a schematic representation of the purificationprotocol protocol for the low molecular weight endoglucanases I and IIfrom Acidothermus cellulolyticus. ##STR2##

What is claimed is:
 1. A substantially purified high molecular weightcellulase enzyme having a molecular weight of between about 156,600 toabout 203,400 daltons from the bacterium Acidothermus cellulolyticus(ATCC 43068), said enzyme is water soluble, possesses both C_(l) andC_(x) types of enzyme activity, a high degree of stability toward heat,and exhibits optimum temperature for activity at about 65° C. at pH'sfrom about 2 to about 9 and an inactivation temperature of about 90° C.at pH's from about 2 to about
 9. 2. The purified cellulose enzyme ofclaim 1, wherein the pH for the optimum temperature activity is fromabout 4 to about 8, and the pH for the inactivation temperature is fromabout 4 to about
 8. 3. The purified cellulase enzyme of claim 1, whereinthe pH for the optimum temperature activity is about 5, and the pH forthe inactivation temperature is about
 5. 4. A method for the productionof the enzyme of claim 1, comprising culturing said bacterium in anutrient medium therefor, filtering crude cellulase enzyme complex byultrafiltration, separating unpurified high molecular weightultrafiltrate fractions by size exclusion chromotography, and loadingsaid fractions on an anion ion exchange column equilibrated againstabout 15 mM Bis-Tris at about pH 6.5 loading buffer, washing saidfractions with said loading buffer, and recovering said high molecularweight cellulase fractions by using a shallow gradient of an increasingionic strength buffer that increases linearly from 0 to about 25 mM ofNaCl, until purified high molecular weight cellulase fractions areeluted off.